Carla António, PhD, leads the Plant Metabolomics Lab at the University of Lisbon, Portugal. She has published extensively on the mass-spectrometry based metabolomics of plant abiotic and biotic stress responses.

• List of Contributors xiiiPreface xix1 Forest Tree Metabolomics Under a Changing Climate 1AnaMargarida Rodrigues and Carla António1.1 Introduction 11.2 Forest Damage 21.3 Forest Tree Metabolomics 51.4 Conclusion and Future Perspectives 72 ExperimentalMethodology for Clonal Forest Research 15Isabel Carrasquinho and Elsa Gonçalves2.1 Introduction 152.2 Defining the Objectives of an Experiment 162.3 Sampling Strategies to Represent the Species 172.4 Planning and Establishing the Experimental Design 172.5 Examples of the Implementation of Field Trials to Quantify Genetic Variability within a Species 192.6 Statistical Analysis and Quantification of Genetic Variability within a Species 252.7 Conclusions 273 Sample Preparation for Forest TreeMetabolomics 35Pia Guadalupe Dominguez, Thaís Regiani Cataldi, Ilka Nacif Abreu, Thomas Moritz, and Ilara Gabriela Frasson Budzinski3.1 Experimental Design for Metabolomics 353.2 Sampling and Quenching of Tree Tissue Material 373.3 Labeling of Tree Tissues 463.4 Metabolite Extraction and Mass Spectrometry-Based Metabolite Analysis 503.5 Conclusions 554 Systems Biology as a Tool to Uncover Interdisciplinary Links within the Complex Forest Tree System 71Pia Guadalupe Dominguez, Ilka Nacif Abreu, ThomasMoritz, and Ilara Gabriela Frasson Budzinski4.1 Systems Biology 714.2 Strategies for Data Integration and Network Analysis 724.3 Integration of Genomics and Metabolomics Data 824.4 Systems Biology to Provide Clues for Metabolite Annotation in Different Tree Species in Recent Years 914.5 Challenges in IntegratingMetabolomics and Other Omics 934.6 Conclusion and Future Perspectives 945 AWorkflow for Metabolomics of Forest Tree Biotic Stress Response and Applications for Management 109Anna O. Conrad, Caterina Villari, and Pierluigi Bonello5.1 Introduction 1095.2 Methods 1115.3 Application 1165.4 Case Studies 1175.5 Conclusions and Future Perspectives 1196 Analysis of Volatile Organic Compounds 127Andrea Ghirardo and Federico Brilli6.1 Plant Volatile Organic Compounds 1276.2 Methodologies for Detecting Plant VOCs 1366.3 Analytical Systems for Measuring Plant VOCs 1406.4 Concluding Remarks and Future Perspectives 1677 Assessing Specialized Metabolites in Tree Bark Using Wide-Targeted LC-MS Analysis 187Maria Kenosis Emmanuelle Galingay Lachica,MutsumiWatanabe, and Takayuki Tohge7.1 Introduction 1877.2 Materials and Methods 1897.3 Data Analysis 1927.4 Data Interpretation 1987.5 Conclusions and Future Perspectives 1998 Plant Hormone Analysis in Forest Tree Species 205Eva Cañizares, Juan Manuel Acién,Miguel González-Guzmán, and Vicent Arbona8.1 Importance of Forest Tree Species 2058.2 Plant Hormones andTheir Roles in Plant Physiology, Biochemistry, and Development 2088.3 Forest Tree Sampling 2138.4 AnalyticalMethods for Plant Hormone Analysis and Profiling 2178.5 Applications of Plant Hormone Profiling to Understand Forest Tree Physiology 2228.6 Future Prospects in Plant Hormone Analysis 2259 Metabolomics of Nutrient-Deprived Forest Trees 235Sara Adrián Lopez de Andrade, Vinícius Henrique de Oliveira, and PauloMazzafera9.1 Introduction 2359.2 Macronutrient Deficiency andWood Production 2379.3 General Use of Mass Spectrometry-Based Metabolomics to Study Wood 2449.4 Tree Nutrition and Metabolome 2489.5 Final Remarks 25310 The Impact of Drought on PlantMetabolism in Quercus Species - From Initial Response to Recovery 267Juan Sobrino-Plata, Francisco Javier Cano, Ismael Aranda, María Brígida Fernández de Simón, and Jesús Rodríguez-Calcerrada10.1 Introduction 26710.2 PrimaryMetabolic Pathways and Metabolite Levels 27610.3 Secondary Metabolic Pathways and Metabolite Levels 28210.4 The Transport of Metabolites within the Plant - Transport Rates and Sap Composition 28510.5 The Release of Metabolites Outside the Plant 28810.6 Conclusions 29311 Metabolomics of Forest Tree Responses to Fluctuations of Temperature and Elevated Atmospheric CO2 315Fernanda Rezende Castro-Moretti, Daniela Feltrim, Sara Adrián Lopez de Andrade, and PauloMazzafera11.1 Introduction 31511.2 Metabolic Response of Trees to Temperature Changes 31811.3 Temperature Effect on Primary Metabolism 31911.4 Temperature Effect on SecondaryMetabolism 32111.5 Effects of Elevated CO2 on Tree Metabolism 32311.6 CO2 Effects on Isoprene Emissions 32711.7 CO2 and Plant Productivity 32711.8 Acclimation After a Long Period of CO2 Exposure 32811.9 The Interactive Effect of Elevated CO2 and High Temperature in Trees 32811.10 Conclusions and Future Perspectives 33112 Integration of Primary Metabolism with Physiological and Anatomical Data to Assess Dutch Elm Disease Susceptibility in Three Elm Species - A Case Study 343Jesús Rodríguez-Calcerrada, AnaMargarida Rodrigues, Carla António, Rosana López, Jorge Domínguez, Juan Sobrino-Plata, Luis Gil, and Juan Antonio Martín12.1 Impacts of Dutch Elm Disease on Plant Metabolism and Its Modulation by Climate 34312.2 Material and Methods 34812.3 Results 35612.4 Discussion 37312.5 Conclusions 37713 Metabolomics of Pinus spp. in Response to Pinewood Nematode Infection 389AnaMargarida Rodrigues, Marta Nunes da Silva, Marta Vasconcelos, and Carla António13.1 Introduction 38913.2 Mass Spectrometry-Based Metabolite Responses to Pinewood Nematode Infection 39113.3 Disease Management 39813.4 Conclusions and Future Perspectives 407References 408Index 421

Carla António, PhD, leads the Plant Metabolomics Lab at the University of Lisbon, Portugal. She has published extensively on the mass-spectrometry based metabolomics of plant abiotic and biotic stress responses.

• List of Contributors xiiiPreface xix1 Forest Tree Metabolomics Under a Changing Climate 1AnaMargarida Rodrigues and Carla António1.1 Introduction 11.2 Forest Damage 21.3 Forest Tree Metabolomics 51.4 Conclusion and Future Perspectives 72 ExperimentalMethodology for Clonal Forest Research 15Isabel Carrasquinho and Elsa Gonçalves2.1 Introduction 152.2 Defining the Objectives of an Experiment 162.3 Sampling Strategies to Represent the Species 172.4 Planning and Establishing the Experimental Design 172.5 Examples of the Implementation of Field Trials to Quantify Genetic Variability within a Species 192.6 Statistical Analysis and Quantification of Genetic Variability within a Species 252.7 Conclusions 273 Sample Preparation for Forest TreeMetabolomics 35Pia Guadalupe Dominguez, Thaís Regiani Cataldi, Ilka Nacif Abreu, Thomas Moritz, and Ilara Gabriela Frasson Budzinski3.1 Experimental Design for Metabolomics 353.2 Sampling and Quenching of Tree Tissue Material 373.3 Labeling of Tree Tissues 463.4 Metabolite Extraction and Mass Spectrometry-Based Metabolite Analysis 503.5 Conclusions 554 Systems Biology as a Tool to Uncover Interdisciplinary Links within the Complex Forest Tree System 71Pia Guadalupe Dominguez, Ilka Nacif Abreu, ThomasMoritz, and Ilara Gabriela Frasson Budzinski4.1 Systems Biology 714.2 Strategies for Data Integration and Network Analysis 724.3 Integration of Genomics and Metabolomics Data 824.4 Systems Biology to Provide Clues for Metabolite Annotation in Different Tree Species in Recent Years 914.5 Challenges in IntegratingMetabolomics and Other Omics 934.6 Conclusion and Future Perspectives 945 AWorkflow for Metabolomics of Forest Tree Biotic Stress Response and Applications for Management 109Anna O. Conrad, Caterina Villari, and Pierluigi Bonello5.1 Introduction 1095.2 Methods 1115.3 Application 1165.4 Case Studies 1175.5 Conclusions and Future Perspectives 1196 Analysis of Volatile Organic Compounds 127Andrea Ghirardo and Federico Brilli6.1 Plant Volatile Organic Compounds 1276.2 Methodologies for Detecting Plant VOCs 1366.3 Analytical Systems for Measuring Plant VOCs 1406.4 Concluding Remarks and Future Perspectives 1677 Assessing Specialized Metabolites in Tree Bark Using Wide-Targeted LC-MS Analysis 187Maria Kenosis Emmanuelle Galingay Lachica,MutsumiWatanabe, and Takayuki Tohge7.1 Introduction 1877.2 Materials and Methods 1897.3 Data Analysis 1927.4 Data Interpretation 1987.5 Conclusions and Future Perspectives 1998 Plant Hormone Analysis in Forest Tree Species 205Eva Cañizares, Juan Manuel Acién,Miguel González-Guzmán, and Vicent Arbona8.1 Importance of Forest Tree Species 2058.2 Plant Hormones andTheir Roles in Plant Physiology, Biochemistry, and Development 2088.3 Forest Tree Sampling 2138.4 AnalyticalMethods for Plant Hormone Analysis and Profiling 2178.5 Applications of Plant Hormone Profiling to Understand Forest Tree Physiology 2228.6 Future Prospects in Plant Hormone Analysis 2259 Metabolomics of Nutrient-Deprived Forest Trees 235Sara Adrián Lopez de Andrade, Vinícius Henrique de Oliveira, and PauloMazzafera9.1 Introduction 2359.2 Macronutrient Deficiency andWood Production 2379.3 General Use of Mass Spectrometry-Based Metabolomics to Study Wood 2449.4 Tree Nutrition and Metabolome 2489.5 Final Remarks 25310 The Impact of Drought on PlantMetabolism in Quercus Species - From Initial Response to Recovery 267Juan Sobrino-Plata, Francisco Javier Cano, Ismael Aranda, María Brígida Fernández de Simón, and Jesús Rodríguez-Calcerrada10.1 Introduction 26710.2 PrimaryMetabolic Pathways and Metabolite Levels 27610.3 Secondary Metabolic Pathways and Metabolite Levels 28210.4 The Transport of Metabolites within the Plant - Transport Rates and Sap Composition 28510.5 The Release of Metabolites Outside the Plant 28810.6 Conclusions 29311 Metabolomics of Forest Tree Responses to Fluctuations of Temperature and Elevated Atmospheric CO2 315Fernanda Rezende Castro-Moretti, Daniela Feltrim, Sara Adrián Lopez de Andrade, and PauloMazzafera11.1 Introduction 31511.2 Metabolic Response of Trees to Temperature Changes 31811.3 Temperature Effect on Primary Metabolism 31911.4 Temperature Effect on SecondaryMetabolism 32111.5 Effects of Elevated CO2 on Tree Metabolism 32311.6 CO2 Effects on Isoprene Emissions 32711.7 CO2 and Plant Productivity 32711.8 Acclimation After a Long Period of CO2 Exposure 32811.9 The Interactive Effect of Elevated CO2 and High Temperature in Trees 32811.10 Conclusions and Future Perspectives 33112 Integration of Primary Metabolism with Physiological and Anatomical Data to Assess Dutch Elm Disease Susceptibility in Three Elm Species - A Case Study 343Jesús Rodríguez-Calcerrada, AnaMargarida Rodrigues, Carla António, Rosana López, Jorge Domínguez, Juan Sobrino-Plata, Luis Gil, and Juan Antonio Martín12.1 Impacts of Dutch Elm Disease on Plant Metabolism and Its Modulation by Climate 34312.2 Material and Methods 34812.3 Results 35612.4 Discussion 37312.5 Conclusions 37713 Metabolomics of Pinus spp. in Response to Pinewood Nematode Infection 389AnaMargarida Rodrigues, Marta Nunes da Silva, Marta Vasconcelos, and Carla António13.1 Introduction 38913.2 Mass Spectrometry-Based Metabolite Responses to Pinewood Nematode Infection 39113.3 Disease Management 39813.4 Conclusions and Future Perspectives 407References 408Index 421

Carla António, PhD, leads the Plant Metabolomics Lab at the University of Lisbon, Portugal. She has published extensively on the mass-spectrometry based metabolomics of plant abiotic and biotic stress responses.

• List of Contributors xiiiPreface xix1 Forest Tree Metabolomics Under a Changing Climate 1AnaMargarida Rodrigues and Carla António1.1 Introduction 11.2 Forest Damage 21.3 Forest Tree Metabolomics 51.4 Conclusion and Future Perspectives 72 ExperimentalMethodology for Clonal Forest Research 15Isabel Carrasquinho and Elsa Gonçalves2.1 Introduction 152.2 Defining the Objectives of an Experiment 162.3 Sampling Strategies to Represent the Species 172.4 Planning and Establishing the Experimental Design 172.5 Examples of the Implementation of Field Trials to Quantify Genetic Variability within a Species 192.6 Statistical Analysis and Quantification of Genetic Variability within a Species 252.7 Conclusions 273 Sample Preparation for Forest TreeMetabolomics 35Pia Guadalupe Dominguez, Thaís Regiani Cataldi, Ilka Nacif Abreu, Thomas Moritz, and Ilara Gabriela Frasson Budzinski3.1 Experimental Design for Metabolomics 353.2 Sampling and Quenching of Tree Tissue Material 373.3 Labeling of Tree Tissues 463.4 Metabolite Extraction and Mass Spectrometry-Based Metabolite Analysis 503.5 Conclusions 554 Systems Biology as a Tool to Uncover Interdisciplinary Links within the Complex Forest Tree System 71Pia Guadalupe Dominguez, Ilka Nacif Abreu, ThomasMoritz, and Ilara Gabriela Frasson Budzinski4.1 Systems Biology 714.2 Strategies for Data Integration and Network Analysis 724.3 Integration of Genomics and Metabolomics Data 824.4 Systems Biology to Provide Clues for Metabolite Annotation in Different Tree Species in Recent Years 914.5 Challenges in IntegratingMetabolomics and Other Omics 934.6 Conclusion and Future Perspectives 945 AWorkflow for Metabolomics of Forest Tree Biotic Stress Response and Applications for Management 109Anna O. Conrad, Caterina Villari, and Pierluigi Bonello5.1 Introduction 1095.2 Methods 1115.3 Application 1165.4 Case Studies 1175.5 Conclusions and Future Perspectives 1196 Analysis of Volatile Organic Compounds 127Andrea Ghirardo and Federico Brilli6.1 Plant Volatile Organic Compounds 1276.2 Methodologies for Detecting Plant VOCs 1366.3 Analytical Systems for Measuring Plant VOCs 1406.4 Concluding Remarks and Future Perspectives 1677 Assessing Specialized Metabolites in Tree Bark Using Wide-Targeted LC-MS Analysis 187Maria Kenosis Emmanuelle Galingay Lachica,MutsumiWatanabe, and Takayuki Tohge7.1 Introduction 1877.2 Materials and Methods 1897.3 Data Analysis 1927.4 Data Interpretation 1987.5 Conclusions and Future Perspectives 1998 Plant Hormone Analysis in Forest Tree Species 205Eva Cañizares, Juan Manuel Acién,Miguel González-Guzmán, and Vicent Arbona8.1 Importance of Forest Tree Species 2058.2 Plant Hormones andTheir Roles in Plant Physiology, Biochemistry, and Development 2088.3 Forest Tree Sampling 2138.4 AnalyticalMethods for Plant Hormone Analysis and Profiling 2178.5 Applications of Plant Hormone Profiling to Understand Forest Tree Physiology 2228.6 Future Prospects in Plant Hormone Analysis 2259 Metabolomics of Nutrient-Deprived Forest Trees 235Sara Adrián Lopez de Andrade, Vinícius Henrique de Oliveira, and PauloMazzafera9.1 Introduction 2359.2 Macronutrient Deficiency andWood Production 2379.3 General Use of Mass Spectrometry-Based Metabolomics to Study Wood 2449.4 Tree Nutrition and Metabolome 2489.5 Final Remarks 25310 The Impact of Drought on PlantMetabolism in Quercus Species - From Initial Response to Recovery 267Juan Sobrino-Plata, Francisco Javier Cano, Ismael Aranda, María Brígida Fernández de Simón, and Jesús Rodríguez-Calcerrada10.1 Introduction 26710.2 PrimaryMetabolic Pathways and Metabolite Levels 27610.3 Secondary Metabolic Pathways and Metabolite Levels 28210.4 The Transport of Metabolites within the Plant - Transport Rates and Sap Composition 28510.5 The Release of Metabolites Outside the Plant 28810.6 Conclusions 29311 Metabolomics of Forest Tree Responses to Fluctuations of Temperature and Elevated Atmospheric CO2 315Fernanda Rezende Castro-Moretti, Daniela Feltrim, Sara Adrián Lopez de Andrade, and PauloMazzafera11.1 Introduction 31511.2 Metabolic Response of Trees to Temperature Changes 31811.3 Temperature Effect on Primary Metabolism 31911.4 Temperature Effect on SecondaryMetabolism 32111.5 Effects of Elevated CO2 on Tree Metabolism 32311.6 CO2 Effects on Isoprene Emissions 32711.7 CO2 and Plant Productivity 32711.8 Acclimation After a Long Period of CO2 Exposure 32811.9 The Interactive Effect of Elevated CO2 and High Temperature in Trees 32811.10 Conclusions and Future Perspectives 33112 Integration of Primary Metabolism with Physiological and Anatomical Data to Assess Dutch Elm Disease Susceptibility in Three Elm Species - A Case Study 343Jesús Rodríguez-Calcerrada, AnaMargarida Rodrigues, Carla António, Rosana López, Jorge Domínguez, Juan Sobrino-Plata, Luis Gil, and Juan Antonio Martín12.1 Impacts of Dutch Elm Disease on Plant Metabolism and Its Modulation by Climate 34312.2 Material and Methods 34812.3 Results 35612.4 Discussion 37312.5 Conclusions 37713 Metabolomics of Pinus spp. in Response to Pinewood Nematode Infection 389AnaMargarida Rodrigues, Marta Nunes da Silva, Marta Vasconcelos, and Carla António13.1 Introduction 38913.2 Mass Spectrometry-Based Metabolite Responses to Pinewood Nematode Infection 39113.3 Disease Management 39813.4 Conclusions and Future Perspectives 407References 408Index 421